The present invention relates to the production, by plants, of recombinant preduodenal lipases, in particular recombinant gastric lipases, and to other polypeptide derivatives of these which have a lipase activity, and to their uses, in particular as functional foods or in pharmaceutical compositions or in enzymatic formulations for agro-alimentary or industrial applications.
Dog gastric lipase (DGL) is a glycoprotein of 379 amino acids (AA) having a molecular weight of about 50 kilodaltons (kDa), which is synthesized in the form of a precursor containing a signal peptide at the amino-terminal (NH2-terminal) end and is secreted by median cells of the mucosa of the fundus of the stomach of the dog (Carrixc3xa8re F. et al., 1991).
Human gastric lipase (HGL) is naturally synthesized in the form of a precursor and is described in the publication by Bodmer et al., 1987. The mature HGL protein is constituted by 379 amino acids. Its signal peptide (HGLSP) is composed of 19 amino acids.
These enzymes belong to a family of lipases called xe2x80x9cpreduodenalxe2x80x9d, some members of which have already been purified and in some cases even cloned (Docherty A.J.P. et al., 1985; Bodmer M. W. et al., 1987; Moreau H. et al., 1988; European Patents no. 0 191 061 and no. 0 261 016).
For a long time it has been taken for granted that hydrolysis of food lipids took place in the small intestine by the action of enzymes produced by the pancreas (Bernard C., 1849).
However, findings have suggested that the hydrolysis of triglycerides could have taken place in the stomach by the indirect means of preduodenal enzymes (Volhard, F., 1901; Shonheyder, F., and Volquartz, K., 1945). These enzymes, and in particular dog gastric lipase, have enzymatic and physico-chemical properties which differentiate them from mammalian pancreatic lipases. These differences between gastric and pancreatic lipases essentially relate to the following points: molecular weight, amino acid composition, resistance to pepsin, substrate specificity, optimum pH of action and stability in an acid medium.
Moreover, in vitro, under certain conditions, it is possible to demonstrate a synergistic action between gastric and pancreatic lipases on the hydrolysis of long-chain triglycerides (Gargouri, Y. et al., 1989).
Several pathological situations (cystic fibrosis, exocrine pancreatic insufficiency) where patients are totally or partly lacking in exocrine pancreatic secretion and therefore in enzymes necessary for hydrolysis of foods (amylases, lipases, proteases) are known. Non-absorption of fats in the intestine, and in particular of long-chain triglycerides, manifests itself by a very significant increase in steatorrhoea in these patients and by a very considerable slowing down in weight increase in young patients. To correct this, porcine pancreatic extracts are administered to these subjects at mealtimes. The therapeutic efficacy of these a extracts could be distinctly improved by co-prescription of DGL due to the specificity of its action on long-chain triglycerides.
The article by Carrixc3xa8re et al. (1991) describes the purification and determination of the NH2-terminal sequence of DGL. A process for extraction of this enzyme from dog stomachs is also described in this publication. This process essentially comprises steeping the stomachs of dogs in an acid medium (pH 2.5) in the presence of water-soluble salts which promote the salting out of lipase in the said medium. The DGL can be purified to homogeneity by stages of filtration over a molecular sieve and ion exchange chromatography. The purified DGL obtained by these processes is a glycoprotein having a molecular weight of 49,000 daltons, 6,000 of which correspond to sugars and 43,000 to the protein part.
Obvious reasons of the difficulties of procurement of the stomachs of dogs prevent any development of this process both in the laboratory and industrially. This results in the need to discover a process which allows production of DGL in a large amount, dispensing with the use of the stomachs of dogs.
The nucleotide and peptide sequences of DGL were determined with the aim of industrial production of DGL by a process using genetic engineering. These works have been the subject of the international application no. WO 94/13816, filed on Dec. 16, 1993.
The process for the production of recombinant DGL described in this international application claims Escherichia coli (E. coli) as the transformed host cell which can produce DGL.
Some difficulties encountered during production of recombinant DGL by E. coli, in particular the need to culture large quantities of E. coli in a fermenter, with high costs, have led to inventors seeking other processes for the production of this DGL.
Mammalian cells are, a priori, more suitable for expression of mammalian genes. However, their use poses problems of maturation of proteins. The enzymatic equipment which realises post-translational maturation differs from one tissue, one organ or one species to another. For example, it has been reported that post-translational maturation of a plasma protein may be different if it is obtained from human blood or if it is produced by a recombinant cell, such as ovarian cells of the Chinese hamster or in the milk of a transgenic animal. Furthermore, the low expression levels obtained with mammalian cells involve cultures in vitro in very large volumes at high costs. The production of recombinant proteins in the milk of transgenic animals (mice, sheep and cows) allows production costs to be reduced and the problems of the level of expression to be overcome. However, ethical problems and problems of viral and subviral contamination (prions) remain.
For these reasons, transgenesis of mammalian genes into a plant cell could provide a route for production of new recombinant proteins in large quantities, at a reduced production cost and without risk of viral or subviral contamination.
In 1983, several laboratories discovered that it was possible to transfer a heterologous gene into the genome of a plant cell (Bevan et al., 1983; Herrera-Estrella et al., 1983 a and b) and to regenerate transgenic plants from these genetically modified cells. All the cells of the plant thus have the genetically modified characteristic, which is transmitted to the descendants by sexed fertilization.
As a result of these works, various teams concerned themselves with the production of mammalian recombinant proteins in plant cells or in transgenic plants (Barta et al., 1986; Marx, 1982). One of the first truly significant results is in this field was the production of antibodies in transgenic tobacco plants (Hiatt et al., 1989).
To express a heterologous protein in the seed, the protein storage site in plants, Vandekerckhove""s team (1989) fused the sequence which codes for leu-enkephalin to the gene which codes for the 2S albumin of Arabidopsis thaliana. With this construction, transgenic rape plants which express the leu-enkephalin specifically in the seeds at expression levels of the order of 0.1% of the total proteins were produced. In 1990, Sijmons and colleagues transferred the gene of human serum albumin into cells of tobacco and potato. Whatever the origin of the signal peptides (human or plant), human serum albumin levels of the order of 0.02% of the total proteins were obtained in potato leaves, stems and tubers.
Other mammalian recombinant proteins have also been produced in plants: the surface antigen of hepatitis B (Mason et al., 1992), interferons (De Zoeten et al., 1989; Edelbaum et al., 1992; Truve et al., 1993); a murine anti-Streptococcus mutans antibody, the agent of dental caries (Hiatt and Ma, 1992; Ma et al., 1994), fragments of the scFV anti-cancer cell antibody (Russel D., 1994), an anti-herpes antibody (Russel D., 1994), hirudin (Moloney et al., 1994), the cholera toxin (Hein R., 1994) and human epidermal growth factor (EGF) (Higo et al., 1993).
All of these researches have demonstrated that the production of mammalian recombinant proteins in plant cells is possible and that the mechanisms of synthesis of proteins from DNA sequences are similar in animal cells and plant cells. However, some differences exist between plant and animal cells, in particular in the maturation of polymannoside glycans into complex glycans, or in the cleavage sites of signal peptides, and it thus cannot be ensured that active or sufficiently active mammalian proteins are obtained by transformation of plant cells.
The inventors have demonstrated that the use of plant cells transformed by an appropriate recombinant nucleotide sequence allows recombinant DGL, or recombinant HGL, or polypeptides derived from these having a sufficient enzymatic activity to be capable of being developed in an industrial application to be obtained.
The aim of the present invention is to provide a new process for the production, by plants, of mammalian recombinant preduodenal lipases, and more particularly of recombinant DGL or HGL, or polypeptides derived from these having an enzymatic activity, and more particularly a lipase activity, such that the said recombinant lipases or their derived polypeptides can be used industrially.
Another aim of the present invention is to provide tools for carrying out such a process, in particular new recombinant nucleotide sequences, genetically transformed plant cells, genetically transformed plants or parts of plant (notably leaves, stems, fruits, seeds or grains, roots) and genetically transformed fragments of these plants or parts of plants.
The aim of the invention is also to provide new mammalian recombinant preduodenal lipase(s), or any derived polypeptide, which are enzymatically active and are obtained from genetically transformed plant cells or plants.
The aim of the invention is also to provide new enzymatic compositions which can be used in the context of carrying out enzymatic reactions, in particular on an industrial scale.
The aim of the invention is also to provide new pharmaceutical compositions, in particular in the context of treatment of pathologies associated with a deficit in the production of lipase in the organism, such as cystic fibrosis.
Another aim of the present invention is to provide new fuels, also called biofuels, which have the advantage of being less polluting than fuels derived from petroleum and of being cheaper to produce.